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PMA/ionomycin treatment (200 ng/mL and 1 μg/mL, respectively – Sigma-Aldrich) was used as a positive control and for staining boundaries (data not shown).
We therefore made clones over-expressing Caps and Trn but they did not show any visible furrow or ommatidial spacing defects at the clone boundaries (data not shown).
Direct comparison of the human and Xenopus genomic sequences revealed perfect conservation of exon/intron boundaries (data not shown).
Notably, an additional member of the FGF superfamily of soluble signals, FGF19, was also shown at hindbrain boundaries (data not shown and see also (Gimeno and Martinez, 2007; Kurose et al., 2004; Weisinger et al., 2010).
japonicus (gi: 17736840) displayed similarity to the entire query sequence, but we decided to omit it from further analyses due to uncertainties in positions of intron-exon boundaries (data not shown).
The sequence of each possible exon was entered into a BLASTN to confirm gene identity and was aligned back to human sequences to confirm the location of exon-intron boundaries (data not shown).
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Notably, addition of only the secondary antibody used for dpERK1/2 detection (anti-rabbit HRP antibody), did not reveal HRP staining at hindbrain boundaries (data not showing).
An identical ATR imaging experiment was carried out using a section of bovine nasal cartilage, which did not show this high intensity feature at the tissue boundary (data not shown).
To corroborate our identification of interrhombomeric boundaries, we compared our 5-HT-immunoreacted or hybridized sagittal sections with equivalent sections with mapped homeobox gene expression patterns found in the Allen Atlas database, thus correlating our data with the relevant molecular boundary landmarks (data not shown).
Notably, staining with another ECM dye, Trichrome, which marks collagen fibers, did not reveal any boundary-specific stain (data not shown), confirming the specificity of the above dyes.
Indeed, when induction takes place once boundary formation has already started (E8.5), none of the observed clones crossed the boundaries (n = 30, data not shown).
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Since I tried Ludwig back in 2017, I have been constantly using it in both editing and translation. Ever since, I suggest it to my translators at ProSciEditing.

Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com